The Pld1Flox allele has loxP sites flanking exons 13-14 (including the catalytic activity-dependent first HKD motif) of the phospholipase D1 gene. Removal of the floxed sequences creates a null allele (see Stock No. 028665). These mice may be useful in studying membrane trafficking, membrane fusion, cancer and neurodegeneration.
Gilbert Di Paolo, Columbia University Medical Center
Phospholipase D1 (Pld1) and its related isoform phospholipase D2 (Pld2) hydrolyze phosphatidylcholine to generate bioactive lipid phosphatidic acid (PA), are implicated in membrane trafficking/membrane fusion, and are elevated/up-regulated in various human carcinomas (including colorectal cancer).
The Pld1Flox allele has loxP sites flanking Pld1 exons 13-14 of the Pld1 gene. Mice homozygous for Pld1Flox are viable and fertile with no reported abnormalities. Following exposure to Cre recombinase, the floxed sequences (including the catalytic activity-dependent first HKD motif) are deleted in the cre-expressing tissues; creating a null allele.
For example, breeding Pld1Flox mice to germline Cre-expressing mice results in the PLD1 global knockout allele Pld1- (available as Stock No. 028665). Those homozygotes (Pld1-/-) are viable and fertile with no reported abnormalities. No protein expression from the knockout allele is detected by western blot analysis of homozygous liver, brain and embryonic fibroblasts.
Furthermore, to study the effect of PLD1-deficiency on spontaneous intestinal tumorigenesis, the Pld1- animals can be bred to ApcMin mice (Stock No. 002020). Specifically, on a C57BL/6J genetic background, PLD1-deficient ApcMin/+ mice have significantly reduced intestinal tumorigenesis and increased survival. The protection is less significant when ApcMin/+ mice are heterozygous for Pld1-. Similar PLD1-deficiency induced protection was observed for an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model.
In contrast, deficiency of the related isoform PLD2 (Stock No. 028668) does not significantly reduce intestinal tumorigenesis in the C57BL/6J-ApcMin model.
The Pld1Flox allele was created by Dr. Gilbert Di Paolo (Columbia University Medical Center). A targeting vector was designed to have a loxP site upstream of exon 13, and a frt-flanked neo cassette with 3' loxP site just downstream of exon 14 of the phospholipase D1 gene (Pld1) on chromosome 3. The construct was electroporated into C57BL/6 x 129/SvJ embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric males were bred with C57BL/6J females to establish the Pld1Flox Neo colony. The donating investigator reports that mice were then bred to the C57BL/6J-congenic FLP deleter strain (Stock No. 005703) for germline removal of the frt-flanked neo.
The donating investigator reported that the Pld1Flox colony was backcrossed to C57BL/6J wildtype mice for at least six generations (and the Flp-expressing transgene was removed) prior to sending males with black coat color to The Jackson Laboratory Repository in 2016 (see SNP results below). Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).
In 2016, a 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. This revealed 6 of 27 markers were not fixed for C57BL/6 allele-type (i.e., still segregating for 129S allele-type markers [6 different chromosomes]). In addition, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in some mice (note the allele-type for C57BL/6N and 129S are the same for these 5 markers). Collectively, these data show segregation on 7 different chromosomes- suggesting that the mice sent to The Jackson Laboratory Repository were incompletely backcrossed onto C57BL/6, and the C57BL/6 genetic background contributions may be a mix of C57BL/6J;C57BL/6N.
|Allele Name||targeted mutation 1.2, Gilbert Di Paolo|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change)|
|Gene Symbol and Name||Pld1, phospholipase D1|
|Strain of Origin||C57BL/6 x 129/SvJ|
|Molecular Note||A loxP site was inserted upstream of exon 13. An FRT-flanked neo cassette with a 3' loxP site was inserted downstream of exon 14. FLP-mediated recombination removed the neo cassette.|
Mice homozygous for Pld1Flox are viable and fertile with no reported abnormalities. When maintaining a live colony, heterozygous mice may be bred together, to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). Alternatively, homozygous mice may be bred together.
When using the Pld1Flox mouse strain in a publication, please cite the originating article(s) and include JAX stock #028664 in your Materials and Methods section.
"MICE" means mouse strains, their progeny derived by inbreeding or crossbreeding, unmodified derivatives from mouse strains or their progeny supplied by The Jackson Laboratory ("JACKSON"). "PRODUCT(S)" means biological materials supplied by JACKSON, and their derivatives. "SERVICES" means projects conducted by JACKSON for other parties that may include but are not limited to the use of MICE or PRODUCTS. "RECIPIENT" means each recipient of MICE, PRODUCTS, or SERVICES provided by JACKSON including each institution, its employees and other researchers under its control. MICE or PRODUCTS shall not be: (i) used for any purpose other than internal research, (ii) sold or otherwise provided to any third party for any use, or (iii) provided to any agent or other third party to provide breeding or other services. Acceptance of MICE, PRODUCTS or SERVICES from JACKSON shall be deemed as agreement by RECIPIENT to these conditions, and departure from these conditions requires JACKSON’s prior written authorization.
MICE, PRODUCTS AND SERVICES ARE PROVIDED "AS IS". JACKSON EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESS, IMPLIED, OR STATUTORY, WITH RESPECT TO MICE, PRODUCTS OR SERVICES, INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, OR ANY WARRANTY OF NON-INFRINGEMENT OF ANY PATENT, TRADEMARK, OR OTHER INTELLECTUAL PROPERTY RIGHTS.
In case of dissatisfaction for a valid reason and claimed in writing by a purchaser within ninety (90) days of receipt of, PRODUCTS or SERVICES, JACKSON will, at its option, provide credit or replacement for the PRODUCT received or the SERVICES provided; JACKSON makes no other representations and this shall be the exclusive remedy of the purchaser. Please note specific policy for live mice.
Consistent with the requirement for a written understanding regarding animal care and use, the JACKSON Animal Care and Use Committee will review the animal care and use protocol(s) associated with any SERVICES to be performed at JACKSON, and JACKSON shall have ultimate responsibility and authority for the care of animals while on site or in JACKSON custody.
In no event shall JACKSON, its trustees, directors, officers, employees, and affiliates be liable for any causes of action or damages, including any direct, indirect, special, or consequential damages, arising out of the provision of MICE, PRODUCTS, or SERVICES, including economic damage or injury to property and lost profits, and including any damage arising from acts or negligence on the part of JACKSON, its agents or employees. Unless prohibited by law, in purchasing or receiving MICE, PRODUCTS, or SERVICES from JACKSON, purchaser or recipient, or any party claiming by or through them, expressly releases and discharges JACKSON from all such causes of action or damages, and further agrees to defend and indemnify JACKSON from any costs or damages arising out of any third party claims.
MICE, PRODUCTS or SERVICES are to be used in a safe manner and in accordance with all applicable governmental rules and regulations.
The foregoing represents the General Terms and Conditions applicable to JACKSON’s MICE, PRODUCTS or SERVICES. In addition, special terms and conditions of sale of certain MICE, PRODUCTS, or SERVICES may be set forth separately in JACKSON web pages, catalogs, price lists, contracts, and/or other documents, and these special terms and conditions shall also govern the sale of these MICE, PRODUCTS and SERVICES by JACKSON, and by its licensees and distributors.
Acceptance of delivery of MICE, PRODUCTS or SERVICES shall be deemed agreement to these terms and conditions. No purchase order or other document transmitted by purchaser or recipient that may modify the terms and conditions hereof, shall be in any way binding on JACKSON, and instead the terms and conditions set forth herein, including any special terms and conditions set forth separately, shall govern the sale of MICE, PRODUCTS or SERVICES by JACKSON.